Abstract

Purpose: Inhibitors of farnesyltransferase
(e.g., R115777) are being developed for therapy and
prevention of various cancers. The efficacy of R115777 [Zarnestra;
(B)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)-methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone]
to prevent the development of lung tumors in mice was determined.

Experimental Design: Female strain A mice (7–8 weeks of
age) were given 100 mg/kg benzo(a)pyrene [B(a)P] by
i.p. injection, and 4 or 14 weeks later, they were given 50 or 100
mg/kg R115777 by oral gavage 5 days/week. The mice were sacrificed 22
weeks after they received the B(a)P.

Conclusions: These results demonstrated that R115777 can
prevent the development of lung tumors in the A/J mouse model, where
tumors routinely have mutations in the Ki-Rasoncogene.

INTRODUCTION

It is well established in the literature that a significant number
of all human cancers, including lung tumors, have a mutation of a
Ras oncogene. Ras proteins are membrane-associated GTPases
that are involved in the regulation of signal transduction pathways
that control cell differentiation, proliferation, and apoptosis
(1)
. In fact, because of their association with a number
of oncogenic murine viruses, the Ras oncogenes were the
first group of oncogenes identified. Although all three Rasoncogenes (Ha, Ki, and N) have been found to be mutated in
different cancers, mutations in Ki-Ras are the most common
in human lung, colon, and pancreatic cancers. Oncogenic mutations of
Ras at codons 12, 13, and 61 have been reported to decrease
GTPase activity, resulting in persistent constitutively GTP-bound
signaling (2)
.

Because of the known involvement of Ras proteins in cancer and their
early isolation, blocking of Ras activity became an early molecular
target for developing therapeutic or preventive drugs (3)
.
There are indeed a number of potential targets for blocking Ras
activity. Thus, one approach is to directly block the interaction of
Ras with one of its downstream elements. For Ras protein to translocate
to the cell membrane and become active, it must be prenylated,
typically farnesylated. Thus, a variety of drug companies and
investigators have taken the approach of developing inhibitors
directed against the enzyme farnesyltransferase, which farnesylates the
Ras proteins, as a method of blocking Ras activity (4, 5, 6, 7)
.
Although the Ras proteins are farnesylated, a number of additional
proteins can be farnesylated as well. One group of
FTIs,3
which includes R115777 [Zarnestra;
(B)-6-[amino(4-chlorophenyl)(1methyl-1H-imidazol-5-yl)-methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-qui-nolinone
(Fig. 1)⇓]
, competes for the CAAX peptide binding site to prevent farnesylation
of the Ras protein and its subsequent translocation to the membrane
(8)
. R115777 has been shown to have antitumor effects in
four different human tumor xenograft models in nude mice
(9)
. R115777 has also entered Phase II clinical trials for
chemotherapy of solid malignancies (10)
. The purpose of
the study reported here was to evaluate the efficacy of R115777 in
preventing the development of lung tumors in strain A mice, which
routinely have mutations in the Ki-Ras oncogene.

Experimental Design.

The experimental design is illustrated in Table 1⇓
. When the mice were 7–8 weeks old, they were given a single i.p.
injection of 100 mg/kg B(a)P in 0.2 ml of corn oil to induce lung
tumors. Starting at 4 or 14 weeks after administration of B(a)P, the
mice were given 50 or 100 mg/kg R115777 by gavage in 0.2 ml of a 20%β
-cyclodextrin solution. The R115777 was administered 5 days/week for
the duration of the experiment. These dose levels of R15777 were chosen
because they were previously reported to be effective in human tumor
xenograft studies in nude mice (9)
.

Mice were weighed weekly for the first 8 weeks of the experiment, and
then they were weighed every 2–4 weeks until sacrifice. Mice were
sacrificed by carbon dioxide asphyxiation at 22 weeks after B(a)P
administration. Thus, they were given R115777 for 8 or 18 weeks. The
lungs were harvested, fixed overnight in formalin, transferred to 70%
alcohol, and evaluated for tumors before embedding in paraffin for
histology.

Histopathological Evaluation.

Lung tumors were classified as being solid/alveolar adenomas, papillary
adenomas, and undifferentiated carcinomas. The criteria used for
solid/alveolar adenoma classification required well-differentiated
cuboidal-shaped cells obliterating at least three contiguous
alveolar spaces (11)
. Papillary tumors were identified as
well differentiated columnar cells with pleomorphic nuclei expanding
within the lung parenchyma as finger-like projections.

PCNA Labeling Index.

To determine the PCNA labeling index, sections of adenomas were
deparaffinized with xylene and placed in 2N HCl at 50°C for 30 min.
Endogenous peroxidase was quenched with 3% hydrogen peroxide (Sigma)
for 30 min. The sections were blocked with diluted horse serum (Vector
Laboratories, Burlingame, CA) for 30 min and incubated with 100 μl of
monoclonal mouse anti-PCNA (dilution, 1:300; Sigma) at room temperature
for 1 h. The sections were washed and incubated with biotinylated
antimouse IgG (Vector Laboratories) for 30 min at room temperature,
followed by incubation with Vectastain ABC KIT reagent (Vector
Laboratories) for 30 min. Stain was developed with
3,3′-diaminobenzidine tetrahydrochloride for 15 min. The slides were
counterstained with hematoxylin. Nuclei of PCNA-labeled cells stained
brown, whereas unlabeled nuclei were blue.

The PCNA-stained sections were analyzed using NIH Image 1.57 software
with threshold density filters to negate user bias. A ×40 objective
lens was used. The labeling index was determined by enumerating
PCNA-positive cells and then evaluating the total number of tumor cells
present within the high-power field, dividing the PCNA-labeled cells by
the total cell count and multiplying by 100. The total number of cells
evaluated per tumor ranged from 600 to 4800 cells, depending on the
tumor size.

Statistical Analysis.

Results are presented as means ± SE and were analyzed by an ANOVA
followed by a Tukey test, with significance indicated by
P < 0.05.

RESULTS AND DISCUSSION

The FTI R115777 was evaluated for its efficacy in preventing
B(a)P-induced lung tumors in mice. The mice showed no signs of
morbidity or toxicity during the experiment; there was no loss of body
weight, no sign of animal malaise, and no change in behavior or
appearance. The 100 mg/kg dose of R115777 decreased the yield of lung
tumors (Fig. 2)⇓
. Tumor multiplicity was 5.0 ± 0.85, 4.5 ± 0.52, and
2.1 ± 0.31 tumors/mouse in mice that received 0, 50, and 100
mg/kg R115777, respectively. Thus, the high dose of R115777 reduced the
yield of lung tumors by 58%, whereas the lower dose of 50 mg/kg did
not significantly reduce the yield of tumors, causing only a 10%
reduction. Histological classification of tumor morphology showed that
the B(a)P-induced lung tumors were predominately solid/alveolar
neoplasms; only one tumor was found to have papillary morphology (data
not shown). We have reported previously (12)
that B(a)P
induces more solid than papillary lung adenomas in strain A mice.

Effect of R115777 FTI on the multiplicity of
lung tumors in strain A mice, week 24. The mice were sacrificed after
24 weeks after administration of B(a)P. Tumors were enumerated during
gross examination at the time of necropsy. The results are the
means ± SE; the asterisk indicates a statistical
significance of P < 0.05.

The PCNA labeling index showed that lung tumors from animals given 100
mg/kg FTI had significantly reduced numbers of proliferating cells
(roughly 60%) when compared with tumors from the untreated control
group (Fig. 3)⇓
. Although 50 mg/kg R115777 did reduce the PCNA labeling index compared
with the control group, it was not statistically significant.

Effect of R115777 on tumor PCNA labeling index.
B(a)P-induced lung tumors reacted with antibodies against PCNA
demonstrate a significant decrease of proliferating cells in tumors
from animals given 100 mg/kg R115777. The results are the means ±
SE. The asterisk indicates a statistically significance
difference (P < 0.05) for the PCNA index in mice
that received FTI.

The development of lung tumors appears to be sensitive to
chemoprevention long after the administration of the initiating
carcinogen. R115777 decreased tumor multiplicity almost 65% when
administered beginning 14 weeks after B(a)P and for only 8 weeks (Fig. 4)⇓
. This finding implies that most of the effect of R115777 is on later
stages of tumor development in this adenoma model. If a significant
portion of the effects of R115777 related to the earlier portions of
the tumor process, then one would expect greater preventive activity
when treatment with the agent was initiated at 4 weeks
versus 14 weeks post-B(a)P. This finding that prevention can
be initiated later during the prevention process is in agreement with a
previous report using a peptidomimetic FTI, FTI-276 (13)
,
and supports the rationale for proposed clinical trials with this agent
in smokers with previously existing lesions. Budesonide has similarly
been shown to be highly effective in preventing lung tumors when
administered starting 10 weeks after vinyl carbamate (14)
.
Thus, the promotional phase of lung carcinogenesis appears to be
sensitive to chemoprevention. The decreased tumor multiplicity and
decreased proliferation rates that we observed in R115777-treated
tumors are consistent with the primary effects of the FTI being on the
growth rate and progression of lung lesions.

Effect of the duration of R115777 treatment on
yield of lung tumors. Mice given 100 mg/kg R115777 by gavage for 8 or
18 weeks were found to have significantly reduced lung tumor
multiplicity when compared with control animals. The results are the
means ± SE; the asterisk indicates statistical
significance of P < 0.05.

The three types of RAS proteins are primarily farnesylated at the CAAX
motif to be transported to the cell membrane. However, the amino acids
adjacent to this motif can alter the affinity of the
farnesyltransferase protein for the Ras protein. Because of the
differing amino acid binding sequences, the farnesyltransferase enzyme
has different affinities for different Ras proteins. The affinity is
relatively low for Ha-Ras and N-Ras, meaning that
these interactions can be readily competed off, whereas the affinity is
much higher for Ki-Ras, meaning that it cannot easily be
competed off. This correlates with known activities of the FTIs as a
class and R115777 in particular. R11577 is profoundly effective against
tumors with mutated Ha-Ras or N-Ras but variably
effective against tumors with Ki-Ras (9)
,
raising the question of whether Ras is the primary target of
the FTIs in tumors with Ki-Ras mutations. In the case of
tumors with Ha-Ras or N-Ras mutation, inhibition
of the farnesyltransferase enzyme is proposed to prevent the binding of
the Ras protein to the plasma membrane. This results in the
down-regulation of downstream pathways including the mitogen-activated
protein kinase pathway and the phosphatidylinositol 3′-kinase/AKT
pathways, which in turn affect cell proliferation and apoptosis.

Virtually all lung tumors induced by B(a)P in strain A mice have
previously been shown to have mutations in codon 12 of the Ki-Rasoncogene (15)
. In a previous study with another FTI,
FTI-276, the percentage of tumors with Ki-Ras mutations was
similarly high in tumors from control and FTI-276-treated mice
(13)
. The question therefore arises whether FTIs,
including FTI-276 and R115777, are working directly against mutated
Ki-Ras or working against a number of other farnesylated
proteins including Rho, nonmutated forms of Ha-Ras or
N-Ras, and so forth. To test this hypothesis, we are
presently testing R115777 in an A/J tumor model using azoxymethane, an
agent that induces few, if any, lung tumors with Ki-Ras mutations.

The present study demonstrates the efficacy of the FTI R115777, an
imidazole analogue that can be administered p.o. and is presently in
multiple Phase II trials for cancer therapy. Furthermore, the present
studies also examined both a true prevention model (R115777 treatment
from weeks 4–22) and a mixed prevention/therapy model (R115777
treatment from weeks 14–22). The similar effects of both models imply
that most or all of the effects of R115777 occur during the
progression/promotion phase of tumor development. Furthermore, the most
likely scenario for Phase II prevention trials would more closely
parallel the prevention/therapy model using former or present smokers.
These results support the potential use of R115777 in such prevention
trials.

Acknowledgments

We thank Janssen Research Foundation for providing R115777.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

↵1 Supported in part by Contract N01-CN-95107 with
the National Cancer Institute.